The present invention relates to novel methods of staining cell compartments, biological components or macromolecules. Specifically, the present invention relates to chimeric polypeptides and nucleic acid constructs encoding same which can be used to selectively stain or highlight cell compartments, biological components or macromolecules.
Various diagnostic and research techniques rely upon specific or non-specific biological staining typically effected using molecules which are either applied to cells or tissues (dyes) or expressed in cells or tissues (reporter genes).
Dyes are typically used for the purpose of highlighting specific cells within a tissue or specific cell components within a cell. Certain dyes, referred to here as vital stains, are capable of staining cells without killing them. Yet, vital stains are not sufficiently selective for highlighting specific cellular components such as specific receptors, cellular organelles or cytoskeletal structures.
Reporter genes, which encode readily assayed proteins, are utilized for marking specific cell components as well as to track expression of exogenous genes in cells. When expressed, fused to a desired partner protein, such as a structural protein, a visual signal is generated which specifically indicates the intracellular fate and position of the partner protein. Among the commonly used reporter genes are those encoding for β-galactosidase, β-glucuronidase, luciferase, and Green Fluorescent Protein (GFP).
GFP and variants thereof (e.g., CFP, YFP) are generally preferred in cases where cellular functions are to be kept unperturbed or when tracking the fate of an expressed protein over a period of time is required. GFP autofluoresces, therefore its use does not require the addition of exogenous signal-producing substrates as required by reporter enzymes such as β-galactosidase, β-glucuronidase or luciferase.
Although the use of reporter gene constructs provides numerous benefits, their generated signal may be, at times, more difficult to detect as compared to fluorescent dyes and as such use of reporter genes is limited to cases where sensitivity or high resolution is not critical. In addition, such proteins often display a limited repertoire of fluorescence spectra.
The present invention provides a novel approach for highlighting specific cell compartments, biological components or macromolecules in a manner that does not disturb cell functions, is highly target selective, and sensitive.